Estrogen promotes innate immune evasion of Candida albicans through inactivation of the alternative complement system.

Candida albicans is a commensal of the urogenital tract and the predominant cause of vulvovaginal candidiasis (VVC). Factors that increase circulatory estrogen levels such as pregnancy, the use of oral contraceptives, and hormone replacement therapy predispose women to VVC, but the reasons for this are largely unknown. Here, we investigate how adaptation of C. albicans to estrogen impacts the fungal host-pathogen interaction. Estrogen promotes fungal virulence by enabling C. albicans to avoid the actions of the innate immune system. Estrogen-induced innate immune evasion is mediated via inhibition of opsonophagocytosis through enhanced acquisition of the human complement regulatory protein, Factor H, on the fungal cell surface. Estrogen-induced accumulation of Factor H is dependent on the fungal cell surface protein Gpd2. The discovery of this hormone-sensing pathway might pave the way in explaining gender biases associated with fungal infections and may provide an alternative approach to improving women's health.

Activation of classical and alternative complement pathways in the pathogenesis of lung injury in COVID-19.

Lung inflammation and damage is prominent in people infected with SARS-Cov-2 and a major determinant of morbidity and mortality. We report the deposition of complement components in the lungs of people who succumbed to COVID-19 consistent with the activation of the classical and the alternative pathways. Our study provides strong rationale for the expansion of trials involving the use of complement inhibitors to treat patients with COVID-19.

Systemic complement activation in anti-neutrophil cytoplasmic antibody-associated vasculitis and necrotizing glomerulonephritis.

AIM: Due to the accumulating evidence of complement activation in anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), we decided to investigate the possibility of systemic complement activation in patients with Necrotizing Glomerulonephritis secondary to AAV. METHODS: Clinical, laboratory and histological findings, and serum levels of complement components, C3a, C5a and Bb fragment of Factor B and C4d, were estimated in patients with AAV and glomerulonephritis, at time of diagnosis, before any treatment had been applied. All patients were treated with the same immunosuppressive protocol and followed up for total 24 months. Twenty age and sex matched healthy individuals served as controls. RESULTS: Serum levels of all complement components were significantly increased in patients, compared to controls; C5a: 19.9(0.02-48) vs 9.06(2.1-16.3)pg/mL, P = .002, Bb: 7.3(0.02-31.4) vs 0.2(0.02-1.6)pg/mL, P < .0001, C3a: 4.7(0.4-7.2) vs 2.4(1.09-5)pg/mL, P = .05 and C4d: 11.6(0.07-70) vs 0.7(0.07-8.2)pg/mL, P = .001, respectively. There was strong correlation between serum Bb levels and eGFR and FFS2009 score at time of diagnosis (r = -.41, P = .002 and r = .41, P = .003 respectively). Also, serum Bb levels were increased in patients with severe interstitial infiltration (P = .04) and focal necrosis (P = .01) on renal biopsy. Serum Bb levels could also predict renal function outcome during the acute phase of disease, but not at the end of follow up. CONCLUSION: We provided strong evidence of systemic activation of complement alternative pathway in the development and progression of AAV and glomerulonephritis. Serum Bb seem to play a critical role in the induction, also predicting disease activity and outcome, yet activation of classical pathway cannot be excluded.

The alternative complement pathway in ANCA-associated vasculitis: further evidence and a meta-analysis.

We compared the common pathway components C3a, C5a and membrane attack complex (MAC), also known as C5b-9, and the alternative pathway components factor B and properdin in patients with ANCA-associated vasculitis (AAV) and healthy controls, and conducted a meta-analysis of the available clinical evidence for the role of complement activation in the pathogenesis of AAV. Complement components were evaluated in 59 patients with newly diagnosed or relapsing granulomatosis with polyangiitis or microscopic polyangiitis and 36 healthy volunteers. In 28 patients, testing was repeated in remission. Next, we performed a meta-analysis by searching databases to identify studies comparing complement levels in AAV patients and controls. A random-effects model was used for statistical analyses. The median concentrations of MAC, C5a, C3a and factor B were higher in active AAV patients (P < 0·001). Achievement of remission was associated with reductions in C3a (P = 0·005), C5a (P = 0·035) and factor B levels (P = 0·045), whereas MAC and properdin levels did not change. In active AAV, there were no effects of ANCA specificity, disease phenotype, previous immunosuppression or disease severity on complement levels. A total of 1122 articles were screened, and five studies, including this report, were entered into the meta-analysis. Plasma MAC, C5a and factor B in patients with active AAV were increased compared to patients in remission (excluding factor B) and controls. Changes in C3a were of borderline significance. Our findings and the results of the meta-analysis support activation of the complement system predominantly via the alternative pathway in AAV patients.

C3 Glomerulopathy: Pathogenesis and Treatment.

C3 glomerulopathy (C3G) is a rare set of kidney diseases with 2 patterns: C3 glomerulonephritis (C3GN) and dense deposit disease. Pathogenesis of both diseases is due to complement dysregulation in the alternative pathway. Acquired or genetic alterations of the regulatory proteins of the complement pathway result in C3G. Although the disease is characterized by low C3 levels in serum and C3-dominant staining by immunofluorescence on biopsy, other disease entities such as infection-related glomerulonephritis and masked monoclonal deposits can present similarly. Both the C3GN and dense deposit disease variants of C3G are progressive and recur in transplanted kidneys. Although no direct treatment is available, complement blockers are either available or in the clinical trial phase. This review will survey the pathogenesis of C3GN and current treatment options.

Clinical relevance of membrane attack complex deposition in children with IgA nephropathy and Henoch-Schönlein purpura.

BACKGROUND: IgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury. METHODS: We conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up. RESULTS: We found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed. CONCLUSIONS: We conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

Complement System: a Neglected Pathway in Immunotherapy.

Approved for the treatment of autoimmune diseases, hematological malignancies, and solid cancers, several monoclonal antibodies (mAb) make use of complement in their mechanism of action. Such an assessment is based on comprehensive investigations that used mouse models, in vitro studies, and analyses from patients at initiation (basal level to highlight deficiencies) and after treatment initiation (mAb impact on complement), which have further provided key insights into the importance of the complement activation and/or complement deficiencies in mAb activity. Accordingly, new approaches can now be developed with the final objective of increasing the clinical efficacy of mAb. These improvements include (i) the concurrent administration of fresh frozen plasma during mAb therapy; (ii) mAb modifications such as immunoglobulin G subclass switching, Fc mutation, or IgG hexamerization to improve the fixation and activation of C1q; (iii) optimization of the target recognition to induce a higher complement-dependent cytotoxicity (CDC) and/or complement-dependant cellular cytotoxicity (CDCC); and (iv) the control of soluble and cellular complement inhibitors.

Diagnosis and treatment of C3 glomerulopathy in a center of expertise.

C3 glomerulopathy is a rare renal disease that has been distinguished as a renal disease for about 10 years. It is caused by an excessive activation of the alternative complement pathway in the circulation, which leads to deposition of complement factor C3 in glomeruli. It is diagnosed based on clinical presentation, histological patterns in a kidney biopsy and tests of the complement pathways. It can closely resemble immune complexmediated glomerulonephritis and postinfectious glomerulonephritis. Renal failure develops in up to half of all patients within 10 years after presentation. A curative treatment is not available. Treatment relies on renoprotective measures, occasional immunosuppressive medication and experimental novel complement inhibitors. Because the disease is rare, its care and cure are concentrated in centers of expertise. Here we provide an overview of the state-ofthe-art diagnosis and treatment of C3 glomerulopathy in a center of expertise in the Netherlands.

Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics.

Anti-CD20 monoclonal antibodies (mAbs) rituximab and ofatumumab are potent activators of the classical complement pathway, and have been approved for the treatment of B-cell malignancies. However, complement exhaustion and overexpression of complement inhibitors by cancer cells diminish their therapeutic potential. The strategies of targeting membrane complement inhibitors by function-blocking antibodies and the supplementation with fresh frozen plasma have been proposed to overcome tumour cell resistance. We present a novel approach, which utilizes gain-of-function variants of complement factor B (FB), a component of alternative C3/C5 convertases, which augment mAb-activated reactions through a positive feedback mechanism called an amplification loop. If complement concentration is limited, an addition of quadruple gain-of-function FB mutant p.D279G p.F286L p.K323E p.Y363A (or selected single mutants) results in significantly increased complement-mediated lysis of ofatumumab-resistant tumour cells, as well as the complete lysis of moderately sensitive cells. Importantly, this effect cannot be achieved by further increasing ofatumumab concentration. Potentiation of cytotoxic effect towards moderately sensitive cells was less apparent at physiological serum concentration. However, an addition of hyperactive FB could compensate the loss of cytotoxic potential of serum collected from the NHL and CLL patients after infusion of rituximab. Residual levels of rituximab in such sera, in combination with added FB, were able to efficiently lyse tumour cells. We suggest that the administration of gain-of-function variants of FB can restore cytotoxic potential of complement-exhausted serum and maximize the therapeutic effect of circulating anti-CD20 mAbs.

Complement Regulation and Immune Evasion by Hepatitis C Virus.

A prominent role for complement has been identified in the linkage of innate and adaptive immunity. The liver is the main source of complement and hepatocytes are the primary sites for synthesis of complement components in vivo. We have discovered that hepatitis C virus (HCV) impairs C4 and C3 synthesis. Liver damage may diminish capacity of complement synthesis in patients. However, we observed that the changes in measured complement components in chronically HCV infected patients do not correlate with liver fibrosis or rheumatoid factor present in the blood, serum albumin, or alkaline phosphatase levels. Complement component C3 is of critical importance in B cell activation and T cell-dependent antibody responses. C3 activity is required for optimal expansion of CD8+T cells during a systemic viral infection. Deficiencies in complement may predispose patients to infections via ineffective opsonization, and defects in lytic activity via membrane attack complex. Interestingly, C9 is significantly reduced at the mRNA level in chronically HCV infected liver biopsy specimens, while many hepatocyte derived complement components (C6, C8, Factor B, MASP1, and MBL) and unrelated genes remain mostly unaffected. This implies an HCV specific effect, not a global effect from liver disease.

C3 glomerulopathy associated with monoclonal Ig is a distinct subtype.

Monoclonal immunoglobulins (MIg) may play a causal role in C3 glomerulopathy (C3G) by impairing regulation of the alternative pathway of complement. Ninety-five patients with C3G were tested for MIg of which 36 were positive. Their mean age at diagnosis was 60 years and among patient 50 years and older, 65.1% had a MIg. At presentation, median serum creatinine and proteinuria were 1.9 mg/dL and 3.0 g/24 hours. Hematuria was present in 32 (88.9%) patients. Twelve (34.3%) patients had low C3 levels. C3 nephritic factor was detected in 45.8% patients; pathogenic variants in complement protein genes were rare. Hematologic evaluation revealed monoclonal gammopathy of renal significance in 26 patients, multiple myeloma in five, smoldering multiple myeloma in two, and chronic lymphocytic leukemia, lymphoma, or type I cryoglobulin each in one patient. After a median follow-up of 43.6 months, the median serum creatinine and proteinuria were 1.4 mg/dL and 0.8g/24 hours. Nine patients developed ESRD. Sixteen patients received MIg-targeted treatment, 17 patients received non-targeted treatment while three patients were managed conservatively. Of the 16 patients receiving MIg-targeted treatment, ten achieved complete/very good/partial hematologic response. Of these, seven achieved a complete/partial/stable renal response. Five patients receiving targeted treatment did not achieve hematologic response, none had a renal response. Patients receiving targeted treatment were more likely to have multiple myeloma/smoldering multiple myeloma. Patients receiving non-targeted treatment were more likely to have monoclonal gammopathy of renal significance. Thus, C3G with MIg is seen in older patients, C3 nephritic factor is the most common autoantibody detected, and MIg-targeted treatment may result in remission and stabilization of kidney function in a subset of these patients.

Recurrent postinfectious glomerulonephritis: an unusual evolution compatible with C3 glomerulopathy.

Acute endocapillary glomerulonephritis, as its name suggests, is a one-time process, which usually resolves within weeks. However, in a small percentage of patients, the disease becomes chronic. In these cases, a deregulation in the alternative complement pathway, which can be caused by mutations or autoantibodies, has been proposed as a pathophysiological mechanism. As a result, the alternative complement pathway remains active after resolution of infection. We report a patient with two episodes of acute renal failure, both times diagnosed by renal biopsy of acute endocapillary glomerulonephritis, with slow recovery after two episodes of low-serum complement C3, haematuria and proteinuria.

C3 glomerulonephritis and dense deposit disease share a similar disease course in a large United States cohort of patients with C3 glomerulopathy.

C3 glomerulonephritis (C3GN) and dense deposit disease comprise the two classes of C3 glomerulopathy. Studies from Europe and Asia have aided our understanding of this recently defined disorder, but whether these data apply to a diverse United States patient population remains unclear. We, therefore, reviewed clinical and histopathological data, including generation of a C3 Glomerulopathy Histologic Index to score biopsy activity and chronicity, to determine predictors of progression to end-stage renal disease (ESRD) and advanced chronic kidney disease (CKD) in 111 patients (approximately 35% non-white) with C3 glomerulopathy: 87 with C3GN and 24 with dense deposit disease. Complement-associated gene variants and autoantibodies were detected in 24% and 35% of screened patients, respectively. Our C3 Glomerulopathy Histologic Index denoted higher activity in patients with C3GN and higher chronicity in patients with dense deposit disease. Over an average of 72 months of follow-up, remission occurred in 38% of patients with C3GN and 25% of patients with dense deposit disease. Progression to late-stage CKD and ESRD was common, with no differences between C3GN (39%) and dense deposit disease (42%). In multivariable models, the strongest predictors for progression were estimated glomerular filtration rate at diagnosis (clinical variables model) and tubular atrophy/interstitial fibrosis (histopathology variables model). Using our C3 Glomerulopathy Histologic Index, both total activity and total chronicity scores emerged as the strongest predictors of progression. Thus, in a large, diverse American cohort of patients with C3 glomerulopathy, there is a high rate of progression to CKD and ESRD with no differences between C3GN and dense deposit disease.

Pathogenic role of inflammatory response during Shiga toxin-associated hemolytic uremic syndrome (HUS).

Hemolytic uremic syndrome (HUS) is defined as a triad of noninmune microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The most frequent presentation is secondary to Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, which is termed postdiarrheal, epidemiologic or Stx-HUS, considering that Stx is the necessary etiological factor. After ingestion, STEC colonize the intestine and produce Stx, which translocates across the intestinal epithelium. Once Stx enters the bloodstream, it interacts with renal endothelial and epithelial cells, and leukocytes. This review summarizes the current evidence about the involvement of inflammatory components as central pathogenic factors that could determine outcome of STEC infections. Intestinal inflammation may favor epithelial leakage and subsequent passage of Stx to the systemic circulation. Vascular damage triggered by Stx promotes not only release of thrombin and increased fibrin concentration but also production of cytokines and chemokines by endothelial cells. Recent evidence from animal models and patients strongly indicate that several immune cells types may participate in HUS physiopathology: neutrophils, through release of proteases and reactive oxygen species (ROS); monocytes/macrophages through secretion of cytokines and chemokines. In addition, high levels of Bb factor and soluble C5b-9 (sC5b-9) in plasma as well as complement factors adhered to platelet-leukocyte complexes, microparticles and microvesicles, suggest activation of the alternative pathway of complement. Thus, acute immune response secondary to STEC infection, the Stx stimulatory effect on different immune cells, and inflammatory stimulus secondary to endothelial damage all together converge to define a strong inflammatory status that worsens Stx toxicity and disease.

C3 glomerulonephritis with a severe crescentic phenotype.

BACKGROUND: C3 glomerulopathy (C3G) is rare type of glomerulonephritis resulting from the glomerular deposition of C3 due to dysregulation of the alternative pathway of complement. It is further subdivided into C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), depending on the ultrastructural features. C3GN usually presents with a membranoproliferative pattern of injury. Crescents may or may not be present. However, we have noted a severe necrotizing and crescentic glomerulonephritis in a small subset of C3GN patients. CASE DIAGNOSIS/TREATMENT: We present the clinical features, kidney biopsy findings, complement evaluation, treatment, and follow-up of four C3GN patients. We also compare the findings with four DDD patients with a severe necrotizing and crescentic glomerulonephritis. The patients were young and presented with hematuria and proteinuria. The biopsy was remarkable for the large number of crescents, in association with bright glomerular staining for C3. The patients were treated with steroids/immunosuppressive drugs. Three of the 4 C3GN patients and 2 of the 4 DDD patients showed stable renal function at follow-up. We find remarkable similarities between our C3GN patients with crescents and the previously described entity of juvenile acute nonproliferative glomerulonephritis. CONCLUSIONS: To summarize, C3G with a severe crescentic phenotype is rare, affects children and young adults, and has a variable response to steroid and immunosuppressive treatment. It is important to recognize this rare cause of crescentic glomerulonephritis so that appropriate evaluation and treatment can be carried out.

The role of the complement system in cancer.

In addition to being a component of innate immunity and an ancient defense mechanism against invading pathogens, complement activation also participates in the adaptive immune response, inflammation, hemostasis, embryogenesis, and organ repair and development. Activation of the complement system via classical, lectin, or alternative pathways generates anaphylatoxins (C3a and C5a) and membrane attack complex (C5b-9) and opsonizes targeted cells. Complement activation end products and their receptors mediate cell-cell interactions that regulate several biological functions in the extravascular tissue. Signaling of anaphylatoxin receptors or assembly of membrane attack complex promotes cell dedifferentiation, proliferation, and migration in addition to reducing apoptosis. As a result, complement activation in the tumor microenvironment enhances tumor growth and increases metastasis. In this Review, I discuss immune and nonimmune functions of complement proteins and the tumor-promoting effect of complement activation.

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 µM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1ß or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1ß had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.

The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy.

Proliferative retinopathic diseases often progress in 2 phases: initial regression of retinal vasculature (phase 1) followed by subsequent neovascularization (NV) (phase 2). The immune system has been shown to aid in vascular pruning in such retinopathies; however, little is known about the role of the alternative complement pathway in the initial vascular regression phase. Using a mouse model of oxygen-induced retinopathy (OIR), we observed that alternative complement pathway-deficient mice (Fb(-/-)) exhibited a mild decrease in vascular loss at postnatal day (P)8 compared with age- and strain-matched controls (P = 0.035). Laser capture microdissection was used to isolate the retinal blood vessels. Expression of the complement inhibitors Cd55 and Cd59 was significantly decreased in blood vessels isolated from hyperoxic retinas compared with those from normoxic control mice. Vegf expression was measured at P8 and found to be significantly lower in OIR mice than in normoxic control mice (P = 0.0048). Further examination of specific Vegf isoform expression revealed a significant decrease in Vegf120 (P = 0.00032) and Vegf188 (P = 0.0092). In conjunction with the major modulating effects of Vegf during early retinal vascular development, our data suggest a modest involvement of the alternative complement pathway in targeting vessels for regression in the initial vaso-obliteration stage of OIR.